Tertiary Structure in Transfer Ribonucleic Acids

136

166

Compact but non-native intermediates have been implicated in the hierarchical folding of several large RNAs, but there is little information on their structure. In this article, ribonuclease and hydroxyl radical cleavage protection assays showed that base pairing of core helices stabilize a compact state of a small group I ribozyme from Azoarcus pre-tRNA ile . Base pairing of the ribozyme core requires 10-fold less Mg 2؉ than stable tertiary interactions, indicating that assembly of helices in the catalytic core represents a distinct phase that precedes the formation of native tertiary structure. Tertiary folding occurs in <100 ms at 37°C. Such rapid folding is unprecedented among group I ribozymes and illustrates the association between structural complexity and folding time. A 3D model of the Azoarcus ribozyme was constructed by identifying homologous sequence motifs in rRNA. The model reveals distinct structural features, such as a large interface between the P4 -P6 and P3-P9 domains, that may explain the unusual stability of the Azoarcus ribozyme and the cooperativity of folding.RNA modeling ͉ RNA structure ͉ metal ions ͉ hydroxyl radical footprinting T he assembly of RNA into functional structures underlies many steps in gene expression and regulation. Recent work has outlined the Mg 2ϩ -dependent folding pathways of large ribozymes (1, 2). Experimental and theoretical results suggest that the initial association of divalent cations induces collapse of the extended RNA chain into more compact structures that favor formation of tertiary interactions (3-8). The structure of the compact intermediates and the extent to which these interactions lead to the native conformation are not yet characterized.On the one hand, individual domains of the Tetrahymena group I ribozyme and the catalytic domain of RNase P fold on a time scale (10-100 ms) similar to the initial collapse transitions of the ribozyme (6, 9, 10). On the other hand, nonspecific collapse results in an ensemble of native and non-native conformations. Many larger RNAs, such as the Tetrahymena group I and Bacillus subtilis RNase P ribozymes, fold slowly in vitro because a large fraction of the RNA population becomes trapped in misfolded intermediates (11,12). An important question is whether RNAs with a simpler 3D architecture are more likely to fold directly to the native structure, as expected from theoretical models (4). Furthermore, we want to know whether the likelihood of misfolding can be predicted from the specificity of counterion-induced collapse.We investigated the Mg 2ϩ -dependent equilibrium folding pathway and constructed a 3D model of the Azoarcus group I intron. The 205-nt group IC3 intron in the pre-tRNA ile of the Azoarcus bacterium is the smallest known self-splicing group I intron (13). It retains the conserved catalytic core common to all group I introns, but lacks the peripheral domains that stabilize folding intermediates of the larger Tetrahymena intron (14).We observed two macroscopic folding transitions in the Azoarcus ribozyme...

Section: D Model Of the Azoarcus Ribozymementioning

confidence: 99%

Assembly of core helices and rapid tertiary folding of a small bacterial group I ribozyme

Rangan

Masquida

Westhof

et al. 2003

136

166

“…Because of their polycationic nature, they bind to the highly negative nucleic acids (1, 2). They have profound effects on protein synthesis in general and on tRNA folding in particular (3,4). The crystal structure analysis of yeast tRNAPhe has been made possible by the observation that the addition of spermine to yeast tRNAPhe produces crystals that yield diffraction patterns of nearly 2-A resolution (5, 6).…”

Section: Introductionmentioning

confidence: 99%

Structural analysis of spermine and magnesium ion binding to yeast phenylalanine transfer RNA.

Quigley¹,

Teeter²,

Rich³

1978

373

222

Refinement of the diffraction data at 2.5Aresolution from orthorhombic crystals of yeast tRNAPhe has poced to t oint where spermine and magnesium ions can elocated in the d rence electron density map. Two spermine molecules are found: one is located in the major groove at one end of the anticodon stem; the other is near the variable loop and curls around phosphate 10 in a region where the polynucleotide chain takes a sharp turn. Four distinct magnesium ions have been identified: one in the anticodon loop, two in the D loop, and one coordinated with phosphates 8, 9, 11, and 12, where the polynucleotide chain is coiled. The conformation of the anticodon stem and loop is stabilized by the cations at the end of the molecule. The positions of these ions may be related to aspects of the biological activity of tRNA. The spermine and magnesium ions appear to be important in maintaining the overall folding of the tRNA molecule. The polyamines are widely distributed in biological systems. Because of their polycationic nature, they bind to the highly negative nucleic acids (1, 2). They have profound effects on protein synthesis in general and on tRNA folding in particular (3,4). The crystal structure analysis of yeast tRNAPhe has been made possible by the observation that the addition of spermine to yeast tRNAPhe produces crystals that yield diffraction patterns of nearly 2-A resolution (5, 6). In contrast, virtually all other tRNA crystals have somewhat disordered crystal lattices (7). The ordered yeast tRNAPhe lattice has made possible the elucidation of the structure of this molecule in both orthorhombic (8) and monoclinic crystal forms (9, 10). We have continued to study the structural details of yeast tRNAPhe in the orthorhombic lattice by the use of refinement procedures. The present refinement of the structure has produced a very good agreement between the observed and calculated data. Consequently, from the differences between the calculated and observed diffraction data, we can visualize the remaining components in the electron density map. This has allowed us to identify portions of electron density that we have interpreted as belonging to spermine and magnesium ions as well as some probable water sites. Two spermine molecules and four magnesium ions have been identified, and several additional tentative sites have been observed. The positions of both the spermine and magnesium in different parts of the tRNA molecule have lead to the conclusion that these cations both play a significant role in stabilizing the three-dimensional folding of the molecule. These observations suggest possible roles for these ions in modifying the biological activity of tRNA molecules during protein synthesis. METHODSRefinement of the yeast tRNAPhe structure had been previously accomplished by a real space refinement procedure in which rigid constraints are applied to the bond angles and bond lengths 64 of the molecular model being refined (11). This procedure produced a reasonable model with a residual error (R value) of 28% a...

“…In the most studied case, that of the tRNAs, it has been found that some form of structural integrity other than the primary sequence of nucleotides must be maintained in order to ensure the biological activity of these molecules (1,2). This is not surprising, since the tRNAs must be able to interact specifically with a myriad of proteins, including those involved in (a) tRNA maturation (i.e., methylases, thiolases, nucleases, and other modifying enzymes); (b) amino-acid activation (the aminoacyl synthetases); and (c) other translational factors (ribosomal proteins, initiation factor 2, and probably others).…”

mentioning

confidence: 99%

Method for predicting RNA secondary structure.

Pipas¹,

McMahon²

1975

107

We report a method for predicting the most stable secondary structure of RNA from its primary sequence of nucleotides. The technique consists of a series of three computer programs interfaced to take the nucleotide sequence of any RNA and (a) list all possible helical regions, using modified Watson-Crick base-pairing rules; (b) create all possible secondary structures by forming permutations of compatible helical regions; and (c) evaluate each structure for total free energy of formation from a completely extended chain. A free energy distribution and the base-by-base bonding interactions of each possible structure are catalogued by the system and are readily available for examination. The method has been applied to 62 tRNA sequences. The total free-energy of the predicted most stable structures ranged from -19 to -41 kcal/mole (-22 to -49 kJ/mole.) The number of structures created was also highly sequence-dependent and ranged from 200 to 13,000. In nearly all cases the cloverleaf is predicted to be the structure with the lowest free energy of formation.We have developed a technique for predicting the secondary structure of RNA from its primary sequence. The method uses thermodynamic and structural criteria to generate all possible secondary interactions in the molecule and evaluates each for free energy. The technique can be used in conjunction with experimental procedures to elucidate the most favorable conformation of a polyribonucleotide chain.The secondary and tertiary structure of RNA is assumed to play an important role in determining the interactions of these macromolecules with proteins. In the most studied case, that of the tRNAs, it has been found that some form of structural integrity other than the primary sequence of nucleotides must be maintained in order to ensure the biological activity of these molecules (1, 2). This is not surprising, since the tRNAs must be able to interact specifically with a myriad of proteins, including those involved in (a) tRNA maturation (i.e., methylases, thiolases, nucleases, and other modifying enzymes); (b) amino-acid activation (the aminoacyl synthetases); and (c) other translational factors (ribosomal proteins, initiation factor 2, and probably others). In addition to their function in translation, some tRNAs have been shown to be involved in the autogenous regulation of certain cistrons and, thus, must also interact with certain transcriptional components (3).Likewise the rRNAs are believed to possess a definite secondary and tertiary structure which serves to govern their interaction with ribosomal proteins (4). The genomes of certain RNA bacteriophages have also been shown to fold owing to secondary and tertiary interactions (5, 6). In these cases too, the evidence demonstrating the functional significance of specific structure is convincing.Studies attempting to demonstrate structure-function relationships in the RNAs have been hindered by a lack of knowledge concerning the exact nature of the structure of 2017 these molecules in solution. The use of c...